cDNA cloning, recombinant expression and characterization of polypetides with exceptional DNA affinity.
Polypeptides remaining tightly associated with isolated genomic DNA are of interest with respect to their potential involvement in the topological organization and/or function of genomic DNA. Such residual DNA-polypeptide complexes were used for raising monoclonal antibodies by in vitro immunization. Screening of a murine lambdagt11 cDNA library with these antibodies released a positive cDNA (MC1D) encoding a 16 kDa polypeptide. The cloned homologous human cDNA (HC1D) was identified in the dbest data base by partial sequence comparison, and it was sequenced full length. The cDNA-derived amino acid sequences comprise nuclear location signals but none of the known DNA-binding motifs. However, the recombinantly expressed proteins show in vitro DNA binding affinities. A polyclonal antiserum to the recombinant MC1D protein immunostains sub-nuclear structures, and it detects a residual 16 kDa polypeptide on western blots of DNA digests. These results support the conclusion that the cloned cDNAs reflect mRNAs encoding one of the chemically-resistant polypeptides which can be detected in isolated genomic DNA by sensitive techniques, e.g. by125Iodine labeling and SDS-PAGE.
Nehls
P
P
German Cancer Research Center, Division Biochemistry of the Cell (0225) and Biomedical Ultrastructure Research Unit (0195), D-69120 Heidelberg, Germany.
Keck
T
T
Greferath
R
R
Spiess
E
E
Glaser
T
T
Rothbarth
K
K
Stammer
H
H
Werner
D
D
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000906:Antibodies; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D010455:Peptides; D011994:Recombinant Proteins